Platelets play a central role in blood clot formation critical to the maintenance of normal homeostasis (Harder et al., 1995). Pathological thrombus formation producing vascular occlusion can result in stroke, myocardial infarction and unstable angina. Platelet aggregation and shape change are induced by ADP released from damaged vessels and red blood cells. ADP also is secreted by platelets from dense granules on activation, potentiating the aggregation response induced by other agents. Three types of purinergic receptors mediate the platelet response to ADP: P2X, an ADP-gated ion channel and two types of G-protein coupled receptors: P2Y1, coupled to increases in intracellular calcium via Gq, and P2T (P2YADP, P2YAC, P2Ycyc, P2TAC), an ADP receptor coupled to inhibition of adenylyl cyclase through Gi (Ralevic and Burnstock, 1998; Kunapuli, 1998; Jantzen et al., 1999; Boeynaerns et al., 2000). Interestingly, the P2T receptor is irreversibly inactivated by active metabolites of the anti-clotting drugs, ticlopidine and clopidogrel (Savi et al., 2000); and patients with a rare heritable clotting disorder lack P2T receptors (Catteneo et al., 1992; Nurden et al., 1995).
In addition, the P2T receptor may have a role in the central nervous system (CNS). ADP receptors with pharmacological properties similar to the platelet P2T receptor have been identified in B10 brain endothelial cells and rat C6 glioma cells (Webb et al., 1996; Boyer et al., 1993). Until recently however, the P2T G-protein-coupled receptor had remained uncloned (Zhang et al., 2001; Hollopeter et al., 2001).
In the CNS, nucleotides and related compounds are often released in concert with other neurotransmitters and act as signal transduction modulators. It has been reported that treatment of astrocytes with P2T selective agonists produces an increase in arachidonic acid release via a pertussis toxin sensitive G-protein-dependent mechanism (Chen and Chen, 1998). ADP-induced arachidonic acid release from astrocytes stimulates glycogenolysis, suggesting that the ADP receptor may be involved in regulation of glycogen metabolism in the CNS. Arachidonic acid acts directly on glial glutamate transporters to inhibit uptake of glutamate (Robinson and Dowd, 1997), further suggesting that glial ADP receptors may regulate neurotransmitter re-uptake. However, the exact role played by the P2T G-protein-coupled receptor in the CNS is not well understood.
It is well established that many medically significant biological processes are mediated by polypeptides participating in cellular signal transduction pathways that involve G proteins and second messengers, e.g., cAMP, IP3 and diacylglycerol (Lefkowitz, 1991). Some examples of these polypeptides include G-proteins and G-protein-coupled receptors themselves (e.g., G-protein-coupled receptor families I, II, III and IV), G-protein-coupled receptors such as those for biogenic amine transmitters (e.g., epinephrine, norepinephrine and dopamine) (Kobilka et al., 1987(a); Kobilka et al., 1987(b); Bunzow et al., 1988), effector polypeptides (e.g., phospholipase C, adenyl cyclase and phosphodiesterase) and actuator polypeptides (e.g., polypeptide kinase A and polypeptide kinase C) (Simon et al., 1991).
One particular pathway of cellular signal transduction is the inositol phospholipid pathway. In this pathway, an extracellular signal molecule (e.g., epinephrine) binds to a G-protein-coupled receptor. The G-protein-coupled receptor (GPCR) subsequently associates with a specific trimeric G-protein, wherein the trimer is comprised of α, β and γ polypeptide subunits. In the GPCR/G-polypeptide associated state, there is an exchange of GDP for GTP at the G-polypeptide α-subunit, resulting in the dissociation of the α-subunit from the β/γ subunits. The GTP bound α-subunit is the active state of the polypeptide. The active α-subunit further activates phospholipase C, which catalyzes the cleavage of PIP2 to IP3 and diacylglycerol (DAG). The IP3 and DAG serve as second messengers in further signal amplification (e.g., Ca2+ release and phosphorylation). Hydrolysis of GTP to GDP, catalyzed by the G-protein itself, returns the G-protein to its basal, inactive form. Thus, following GPCR binding a ligand molecule, the ligand activates a G-protein. The G-protein serves a dual role, as an intermediate that relays the signal from receptor to effector, and as a clock that controls the duration of the signal.
GPCRs are membrane bound polypeptides, comprising a gene superfamily characterized as having seven putative transmembrane domains. GPCRs can be intracellularly coupled by heterotrimeric G-proteins to various intracellular enzymes, ion channels and transporters (see, Johnson et al., 1989). Different G-protein α-subunits preferentially stimulate particular effectors to modulate various biological functions in a cell.
The G-protein-coupled receptors include a wide range of biologically active receptors, such as hormone receptors, viral receptors, growth factor receptors and neuroreceptors. Examples of members of this family include, but are not limited to, dopamine, calcitonin, adrenergic, endothelin, cAMP, adenosine, muscarinic acetylcholine, serotonin, histamine, thrombin, kinin, follicle stimulating hormone, opsins, endothelial differentiation gene-1, rhodopsins, odorant, and cytomegalovirus receptors.
The seven transmembrane domains are believed to represent transmembrane α-helices connected by extracellular or cytoplasmic loops. GPCRs have been characterized as including these seven conserved hydrophobic stretches of about 20 to 30 amino acids, connecting at least eight divergent hydrophilic loops. Most GPCRs (also known as 7TM receptors) have single conserved cysteine residues in each of the first two extracellular loops which form disulfide bonds that are believed to stabilize functional polypeptide structure. The 7 transmembrane regions are designated as TM1, TM2, TM3, TM4, TM5, TM6, and TM7. TM3 has been implicated in several GPCRs as having a ligand binding site, such as the TM3 aspartate residue. TM5 serines, a TM6 asparagine and TM6 or TM7 phenylalanines or tyrosines are also implicated in ligand binding in certain receptor families.
Phosphorylation and lipidation (palmitylation or farnesylation) of cysteine residues can influence signal transduction of some GPCRs. Most GPCRs contain potential phosphorylation sites within the third cytoplasmic loop and/or the carboxy terminus. For several GPCRs, such as the β-adrenoreceptor, phosphorylation by polypeptide kinase A and/or specific receptor kinases mediates receptor desensitization.
Presently, more than 800 GPCRs from various eukaryotic species have been cloned, 140 of which are human GPCRs for which endogenous ligands are known (Stadel et al., 1997). In addition, several hundred therapeutic agents targeting GPCRs such as angiotensin receptors, calcitonin receptors, adrenoceptor receptors, serotonin receptors, leukotriene receptors, oxytocin receptors, prostaglandin receptors, dopamine receptors, histamine receptors, muscarinic acetylcholine receptors, opioid receptors, somatostatin receptors and vasopressin receptors have been successfully introduced onto the market for various indications (see Stadel et al., 1997). This indicates that these receptors have an established, proven history as therapeutic targets. The search for GPCR genes has also identified numerous genes whose products are members of the GPCR family, but for which their natural ligands are not known, commonly refered to as orphan receptors. In fact, more than 100 of the 240 human GPCRs identified (i.e., about 45%) are orphan receptors, and it is estimated that there are at least 400–1000 more GPCR genes that have yet to be identified (Stadel et al., 1997).
Thus, there is clearly a need for the identification and characterization of further GPCRs, their genes and their ligands, which can play a role in preventing, ameliorating or correcting dysfunctions or diseases, including, but not limited to, infections such as bacterial, fungal, protozoan and viral infections, particularly infections caused by HIV-1 or HIV-2; pain; cancers; anorexia; bulimia; asthma; Parkinson's disease; acute heart failure; hypotension; hypertension; platelet formation and aggregation; stroke; urinary retention; osteoporosis; angina pectoris; myocardial infarction; ulcers; asthma; allergies; benign prostatic hypertrophy; and psychotic and neurological disorders, including anxiety, schizophrenia, manic depression, delirium, dementia, severe mental retardation and dyskinesias, such as Huntington's disease or Gilles dela Tourett's syndrome.